Gel electrophoresis is used to separate proteins and nucleic acids and is one of the most important tools in modern biotechnology. It consists of gel, buffer, and electrodes. Samples are loaded in the gel, electric current is applied to the gel through the buffer from the electrodes, and the samples are separated in the gel according to their mobility difference in electric field. Based on its geometry, gel electrophoresis can be classified as capillary gel electrophoresis, column gel electrophoresis, and slab gel electrophoresis. Slab gel electrophoresis is most widely used among them.
Slab gel electrophoresis utilizes a gel slab as separation media and the gel slab should be separated from its casting mold after electrophoresis for analysis. It is convenient, economic, and simple. Multiple samples can be analyzed at the same time and results can be visualized directly. Slab gel electrophoresis includes two major types, horizontal slab gel electrophoresis and vertical slab gel electrophoresis.
Most agarose gel slabs are horizontal type and are used for analysis of nucleic acids. A horizontal agarose gel slab is prepared in a tray with a flat bottom. Hot agarose solution is poured into the tray and a comb is placed on the tray with its teeth extending in the solution but not touching the bottom of the tray. After the agarose solution solidifies, the comb is removed to leave wells for loading samples. The gel slab is then submerged in a buffer, samples are loaded into the wells, and electric current is applied onto the gel slab to separate the samples. After electrophoresis the gel slab is stained and the sample bands are visualized and photographed.
Horizontal agarose gel electrophoresis becomes an indispensable tool in molecular biology. The agarose gel separates nucleic acids according to size difference with a wide separation range (from dozens of base pairs to millions of base pairs). The slab geometry allows simultaneous analysis of multiple samples. The horizontal format provides a very simple procedure for gel preparation. Horizontal agarose gel electrophoresis is used in gene mapping, library screening, southern/northern blotting, and other researches related to nucleic acid analysis.
Horizontal agarose gel electrophoresis also has some unavoidable disadvantages. Submerging an agarose gel slab in a buffer provides good heat exchange between the gel slab and the buffer, but it also increases the cross section of electric field during electrophoresis, generating high electric current which prevents applying high voltage onto the gel slab. Since mobility of samples in a gel slab is proportional to voltage applied, horizontal slab gel electrophoresis needs long time to complete. Submerging an agarose gel slab in a buffer also prevents from using thin agarose gel slabs because samples in thin gel slabs will easily diffuse into the surrounding buffer and decreases detection sensitivity. The way of forming sample wells on a horizontal agarose gel slab also requires thick gel slab since the bottom of sample wells should be a gel layer of certain thickness to prevent samples from leaking. Sample wells should also be deep enough to load certain volume of samples. Practically, the thickness for a horizontal agarose gel slab is about 5 millimeter.
The thickness of the gel slab adds more disadvantages to horizontal agarose gel electrophoresis. It wastes expensive agarose. It dilutes samples, decreasing sensitivity. It slows down staining/destaining process, increasing analysis time. As a gel slab becomes thick, electric field on the cross section of the gel slab is not uniform which results in band-bending, a major factor for inferior resolution for horizontal agarose gel electrophoresis. It is more problematic for blotting process since a thick gel often results in smear bands and also requires long blotting time.
The submerging nature of a horizontal gel slab prevents it from application of isotachophoresis, a technique used in capillary zone electrophoresis for sample concentration. The technique can increase detection sensitivity by 1 to 2 order of magnitude, making the detection of low abundant components in the sample possible. However, the technology requires the sample loading side and its opposing side of the separating media expose to two different buffers. In horizontal agarose gel electrophoresis, the gel slab is submerged in a single buffer, restricting application of isotachophoresis. In another words, horizontal agarose gel electrophoresis cannot detect low abundant components, greatly downgrading its usefulness.
Contrary to the horizontal agarose gel slab which is free from casting mold during electrophoresis, a vertical gel slab is cast in a detachable cassette which remains integrated with the gel slab during electrophoresis. The detachable cassette consists of two plates, a comb of multiple teeth, and a pair of spacers the two spacers are held between the two plates along the two vertical edges of the two plates, forming a cassette with a rectangular interstice. The bottom of the cassette is detachably sealed and a gelable solution is added into the interstice from its top opening. The comb is placed on the top of the cassette with its teeth extending into the gelable solution. After the gelable solution solidifies, the comb is removed to generate sample wells. The gel-containing cassette is clamped onto a vertical electrophoresis device with the sample loading end and its opposing end exposing to two separate buffer chambers for electrophoresis.
Most of vertical gel slabs are polyacrylamide type and are mainly used for protein analysis and DNA sequencing. The vertical gel slab is protected within the cassette and its two ends expose to two different buffer chambers. Though not reported previously, this configuration allows applying isotachophoresis on the gel to concentrate samples. The vertical cassette format allows preparation of a very thin gel slab for electrophoresis. The thin gel slab provides good heat exchange and has low electric current due to smaller cross-section of electric field so that high voltage can be applied on it to decrease analysis time. The thin gel slab minimizes sample diffusion and results in good resolution. The thin gel slab increases productivity by shortening staining/destaining process. The thin gel slab also saves expensive gel materials. However, an agarose gel slab cannot be easily and effectively prepared in thin vertical format. Most biochemistry laboratories have to keep both horizontal electrophoresis apparatus for agarose gel and vertical electrophoresis apparatus for polyacrylamide gel.
Many factors prevent from preparation of cassette-type vertical agarose gel slabs. To prepare a cassette-type vertical agarose gel slab, hot agarose solution should be added into the narrow cassette interstice. The gel solution solidifies in the cassette when it cools down. Unfortunately, the gel also shrinks during cooling, a severe problem for preparation of a vertical agarose gel slab. Gel shrinkage makes the gel slab thinner and generates void between the cassette plates and the gel slab. A component in a sample travels both in the gel slab and in the void with different mobility, resulting in band diffusion. The cassette has less contact with the thinner gel slab which often slips off from the cassette during electrophoresis gel shrinkage also results in unpredictable gel break. Lowering temperature of agarose solution will reduce the degree of shrinkage. But in this way, the agarose solution will solidify before it reaches the bottom of the interstice generating air bubbles which ruins the gel slab. An agarose gel slab is tender than a polyacrylamide gel slab and deforms when it is clamped onto a vertical electrophoresis apparatus. In general, vertical agarose gel electrophoresis is impractical in the currently available vertical gel electrophoresis systems.
Besides that it is not suitable for agarose gel electrophoresis, the conventional vertical gel electrophoresis system also has other limitations. Since the gel cassette is installed for electrophoresis by clamping its two vertical sides onto an electrophoresis device, the cassette plates have to be thick enough so that they will not bend horizontally by clamping. When a wider cassette is used for loading more samples, thicker plates have to be selected to avoid horizontal bending, which slows down heat exchange. Sample loading capacity is restricted by this factor. Conventional plates for vertical gel electrophoresis are 1 millimeter thick and 80 millimeter wide for mini-gels and 3 millimeter thick and about 300 millimeter wide for sequencing gels.
The spacers on the two vertical sides of the cassette often affect uniformity of the electric field in a vertical gel slab. The sides and the mid-portion of the cassette usually subject to different voltage drop which generates uneven migration of sample bands called "smiling effect". This effect will be maximized by insufficient heat exchange. Smiling effect will affect the quality of electrophoresis and some times makes identification of adjacent bands difficult.
As the size of a cassette increases, preparation of a gel slab in the cassette turns to be difficult. For example, to prepare a DNA sequencing gel slab of 0.1 to 0.4 millimeter thickness in a vertical cassette is very difficult and often fails due to air bubbles generated during addition of the gelable solution. To separate the gel slab from the casting plates is also troublesome and the gel slab often breaks during this process. For this reason, a toxic silanization reagent is spread onto one plate to help the separation of the gel slab from the casting plates. Besides exposure to toxic material, this process can not completely prevent the gel slab from break and skillful workers are still required.
J. Hejgaard reported a hollow cylindrical gel slab for vertical gel electrophoresis the casting mold comprises hollow cylinders A and B. The internal diameter of cylinder A is slightly larger than the outer diameter of cylinder B so that an interstice forms between the two cylinders when cylinder B is inserted into cylinder A. A gel slab forms in the interstice. Although a hollow cylindrical slab gel of 2.5 millimeter thickness was prepared, it has never become practical due to its messy and awkward operation. Unlike the cassette-type vertical gel format in which plates are removed from the gel slab by lifting, one has to pull one cylinder away from the other to expose the gel and the gel slab with the thickness of 1 millimeter or less is unavoidably damaged.
In general, further advancement in slab gel electrophoresis is needed to overcome the limitations of the conventional technologies. There is a need to develop a vertical agarose gel electrophoresis system so that isotachophoresis technique can be applied to agarose gel slabs for higher sensitivity. There is a need to prepare thinner agarose gel for better resolution, shorter analysis time, and lower material consumption. There is a need to increase loading capacity of the current cassette-type vertical gel electrophoresis system without slowing down heat exchange and increasing the physical size of the system. There is a need to overcome the ununiform electric field across the cassette-type vertical gel slab so that smiling effect can be eliminated and better results obtained. There is a need to further simplify the process of vertical slab gel electrophoresis.